DESCRIPTION: The overarching goal of this project is to develop Node-Pore Sensing (NPS), an innovative label-free microfluidic technique, such that it could go beyond flow cytometry in terms of number of markers that can be practically and routinely screened simultaneously and in a non-destructive manner. Currently, flow cytometry is practically limited to 6-10 markers due to spectral emission overlap of the different fluorochromes used simultaneously. NPS measures the transit time of a cell as it interacts (specifically or non-specifically) with antibodies functionalized in a microfluidic channel that has been segmented by nodes. Specific interactions between cell-surface receptors and the functionalized antibody retard the cell, leading to longer transit times and subsequent determination of a particular surface-marker presence. This high-risk, high-reward R21 project has two Specific Aims: Aim 1: To optimize device coding and processing for high throughput screening and real-time analysis. We will design and develop a unique NPS platform based on Barker codes that enable high- resolution detection even with low signal-to-noise ratios (SNRs). Barker codes are binary signals that are often used in radar and telecommunications to which NPS is analogous. Aim 2: To incorporate sorting technology onto the NPS platform developed in Aim 1. We intend to realize the full potential of NPS and integrate a sorting technology to the NPS platform. The sorting technology will be based on mechanical pressure actuation to sort cells rapidly into phenotypic sub- populations for downstream analysis and/or culture. NPS development and proof-of-principle will be based on screening and sorting breast-cancer cell lines, MCF10A, MCF-7, MDA-MB-436, and MDA-MB-231-all of which have different malignancy and metastatic status-for markers EpCAM, CD44, CD24, CD29, CD49f, CD133, Axl, MUC1, EGFR, and ErbB2. By focusing on these markers, we would have an immediate impact in studies involving characterizing sub-populations of circulating tumor cells from patients with metastatic breast cancer. Thus, our proof-of-principle for a fully developed NPS has high impact. The proposed integrated multi-marker NPS and sorting technology proposed has potential for transformative impact in a number of fields ranging from fundamental life sciences research to point-of-care diagnostics. For example, flow cytometry is the cornerstone to diagnosis for many of the hematologic malignancies. With our screening/sorting technique, we could detect minimal residual disease and remission states. Beyond clinical diagnosis, our technology could be employed to characterize, for instance, changes in surface-marker expression during stem-cell differentiation in order to identify and isolate potentially important and rare sub-populations.